Method for determining the fructosamine content

Abstract: Reagents and methods for determining the fructosamine content in a blood sample or a sample derived from blood are disclosed. It is proposed: a set of reagents for determining the fructosamine content in a blood sample or a sample derived from blood, comprising two stable ready to use liquid reagents, the first liquid reagent having an nearby neutral pH and containing a colour reagent, which is reduced by fructosamine under alkaline pH conditions, thereby changing the colour, and reagents for removing non specifically reducing and/or turbidity-causing components of the sample, as well as possibly other usual additives, and the second liquid reagent containing a basic buffering agent, a method of determining the content of fructosamine in a blood sample or a sample derived from blood which uses the above set of reagents.

Description: The invention relates to a method of determining the fructosamine content in a blood sample or a sample derived from blood, by treating the sample with a colour reagent and measuring the induced change of colour, whereby non specifically reducing and/or turbidity-causing components of the sample are removed during the colour reaction, wherein that a first liquid reagent having a pH between 6 and 9 and containing an appropriate non reducing buffering agent, a colour reagent, which is reduced by fructosamine under alkaline pH conditions, thereby changing the colour, and reagents for removing non specifically reducing and/or turbidity-causing components of the sample, is mixed with a second liquid containing a basic buffering agent, and, simultaneously or subsequently, the colour reaction is started by adding the sample, whereby the second liquid reagent is used in such an amount as to adjust the pH of the reaction mixture between 9 and 12. The expression "fructosamine content" means the total content of non enzymatic glycosylation stably attached to proteins. This parameter, which reflects an average blood glucose levels over a period of time, is useful for monitoring metabolic control in diabetes. The expression "non specifically reducing components" means here blood components other than fructosamine which, under alkaline pH conditions, are capable of reducing the colour reagent. Such components may be bilirubin, uric acid and proteins with free thiol groups, as well as vitamins such as ascorbic acid and pharmaceutical metabolites, such as 2,5-dihydroxybenzoic acid, a metabolite of acetylsalycilic acid. The expression "turbidity-causing components" means non watersoluble blood components such as lipids, which are capable of causing turbidity in samples. European Patent Publication No. 85 263 describes a simple method of determining the fructosamine content in serum samples. This method is based on the fact that fructosamines in alkaline solution form eneaminols which act as reducing agents on tetrazolium salts, such as for instance nitro-blue tetrazolium (NBT), thereby causing a change in colour which can be photometrically determined, the measured change in colour in a defined time interval being proportional to the quantity of the present fructosamines. This method however has a number of drawbacks. Problems may arise in particular when using serum as sample material, because other serum components, such as bilirubin, uric acid or proteins with free thiol groups also act as reducing agents on the colour reagents. Besides, lipids, when present in high concentration, may cause turbidity of the sample, which has negative effects on photometric measurements and makes determination of the fructosamine content difficult, if not impossible. Japanese Patent Publication No. 4 328 466 describes a method for determining the serum fructosamine content in blood samples, which comprises (a) treating the sample with an alkaline solution containing a metal ion to remove interfering substances, (b) adding a neutral or acidic solution containing a colour reagent and (c) measuring the resulting change in colour at pH 9.5 to 10.5. The alkaline solution containing a metal ion used in step (a) is taught to comprise a carbonate buffer containing glycine and cobalt sulfate, and a sodium dihydrogenphosphate solution containing 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride. To overcome these drawbacks, it has been proposed in European Patent Publication No. 309 882 to treat the sample before the colour reaction at a nearby neutral pH with reagents, which remove the interfering sample components, and then to set a basic pH, which is necessary for the colour reaction, and start the colour reaction by adding the colour reagent. This process is practically carried out with a set of two distinct ready to use liquid reagents, the first reagent being an almost neutral buffer, which contains the reagents for removing the interfering sample components, and the second reagent being an alkaline buffer, which contains the colour reagent. In order to use this process on automated clinical analysers, the colour reagent and the alcaline buffer must have a relatively high concentration. It has been found however that the colour reagents, usually tetrazolium salts, are not stable in alkaline medium in the presence of relatively high concentrations of colour reagent and buffer salts, so that said second reagent is not shelf-stable. In commercially available kits (Fructosamine Plus® and Unimate Fructosamine® , Hoffmann-La-Roche, Basel, Switzerland), this disadvantage has been avoided by separating the reagents as follows: the first reagent consists of a tablet, which contains the colour reagent and reagents for removing non specifically reducing sample components, such as uricase; the second reagent consists of an alkaline buffer which contains the remaining reagents. In order to make the ready to use solution necessary for the analysis, the tablet (first reagent) must then first be dissolved in the second reagent. This way of proceeding gives excellent results but has following drawbacks: dissolution of the tablet takes at least 15 minutes and cannot be automated; the thus obtained solution of mixed reagents has a usable life time of less than two weeks. There is therefore a need for a method for determining the fructosamine content which do not have the above drawbacks. The problem to be solved by the present invention is to provide such methods. This problem is solved by providing the above method. The colour reagent is a substance capable of being reduced under alkaline pH conditions by the eneaminol form of fructosamine, the reduced form of the colour reagent having a colour different from that of the non reduced form, so that appearance of the reduced form can be photometrically followed. Numerous such colour reagents are found among tetrazolium salts, which in alkaline medium are capable of being reduced to formazans : examples of such tetrazolium salts are: 3,3'-(3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl)-bis(2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride) (nitro-blue tetrazolium NBT), 3,3'-(3,3'-dimethoxy-(1,1'-biphenyl)-4,4'-diyl)-bis(2,5-bis(p-nitrophenyl)-2H-tetrazolium chloride) (TNTB), 3,3'-(1,1'-biphenyl-4,4'-diyl)-bis(2,5-diphenyl-2H-tetrazolium chloride) (NeoTB), 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and 2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium chloride (TV). Tetrazolium salts are preferably used at a concentration between 0.2 and 2 mmol/l in the first liquid reagent. A particularly convenient tetrazolium salt is nitro-blue tetrazolium, which is advantageously used at a concentration between 0.5 and 1.5 mmol/l in the first liquid reagent. Reagents for removing non specifically reducing components are preferably enzymes with oxidizing activity such as uricase, bilirubinoxidase, peroxidase, catalase and/or ascorbic acid oxidase. These enzymes may be stabilized by addition of salts such as potassium chloride. They are advantageously used at a concentration between 10 and 10 7 U/l in the first liquid reagent. Because blood usually contains a substantial amount of uric acid, the latter being the end product of metabolism of purine in man and other primates, uricase, an enzyme which catalyses oxidation of uric acid into allantoin, is especially useful as a reagent for removing non specifically reducing components. Examples of appropriate commercially available uricases are those isolated from Candida albicans (Seppim, Sées, 61500, France), Aspergillus flavus (Merck), pig liver (Serva, Heidelberg,FRG), Arthrobacter globiformis (Sigma), and Arthrobacter protophormiae (Boehringer Mannheim). A particularly interesting uricase is uricase of Arthrobacter protophormiae, which is preferably used at a concentration between 500 and 1500 U/l in the first liquid reagent. Further addition of thiol-blocking agents which react with free thiol groups may greatly improve results. Such thiol-blocking agents are for instance iodoacetamide, iodoacetate, N-ethylmaleinamide and mercuric p-hydroxybenzoate. These reagents are preferably added at a concentration between 0.5 and 500 mmol/l, particularly between 5 and 50 mmol/l, in the first liquid reagent. The preferred thiol-blocking agent is iodoacetamide. Reagents for removing turbidity-causing serum components are enzymes which catalyse their degradation, such as lipases, as well as anionic, cationic and non ionic detergents, if desired in combination with salts of strong acids. Advantageous anionic detergents are salts of bile acids and their conjugates with alkali metals and alkaline-earth metals. Anionic detergents are preferably used at a concentration between 2 and 10 mmol/l in the first liquid reagent. An interesting such salt is sodium cholate, particularly when used at a concentration between 8 and 16 mmol/l in the first liquid reagent. A large variety of non ionic detergents may be used. Proper non ionic detergents include in particular linear and branched alkyl- or arylalkyl-alcohol-polyglycolethers with 8 to 12 carbon atoms in the alcohol part and 4 to 15 glycol units per molecule. Examples of such commercially available non ionic detergents are Genapol X-80 ( an isotridecylpoly(ethylenglycolether)n with n=8, available from Boehringer Mannheim), Oxetal ID 104 (Zschimmer & Schwarz, Lahnstein, FRG), Lutensol ON 50, ON 60, ON 70 (BASF) and Soprofor D/916 (Rhone Poulenc, France) One of such non ionic detergents or a mixture thereof is preferably added at a concentration between 0.05 and 15 % in weight, particularly between 0.1 and 5 % in weight, in the first liquid reagent. Isotridecylpoly(ethyleneglycolether)n is advantageously used, particularly at a concentration between 50 and 110 mmol/l in the first liquid reagent. The effect of removing turbidity-causing components is stronger in the presence of salts of strong acids, such as, for example, salts of sulfuric acid or hydrochloric acid with an alkali metal or an alkaline-earth metal. Preferred such salts are potassium and sodium chloride. Strong acid salts are preferably used at a concentration between 20 and 200 mmol/l in the first liquid reagent. The first liquid reagent having an almost neutral pH contains an appropriate non reducing buffering agent, usually at a concentration between 10 and 100 mmol/l. This buffering agent is advantageously chosen among buffers having a pH between about 6 and about 9, particularly between about 7 and about 8. A particularly convenient buffering agent is potassium phosphate buffer having a pH between about 7 and about 8. The second liquid reagent contains a buffering agent having a pH between about 9 and about 12.5, preferably between about 10 and about 11. An advantageous buffering agent is potassium carbonate buffer having a pH between about 10 and about 11. The above described set of reagents has an excellent stability under conditions of storage and of use. The invention relates to a method of determining the fructosamine content in a blood sample or a sample derived from blood, by treating the sample with a colour reagent and measuring the induced change of colour, whereby non specifically reducing and/or turbidity-causing components of the sample are removed during or prior to the colour reaction, which method uses the above described set of reagents. In an embodiment of this method, the sample is first mixed with the above described first reagent in order to remove the interfering components, then the colour reaction is started by adding an appropriate quantity of the above described second liquid reagent, so as to adjust the pH of the reaction mixture between about 9 and about 11, preferably between about 10 and about 11. The rate of change of colour of the reaction mixture is photometrically determined at an appropriate wavelength and compared to that of a calibrating solution. Advantageous calibrating solutions are those containing a known concentration of ketoamine, preferably glycated poly-L-lysine (cf. EP 351 790), or deoxymorpholinofructose (DMF), or a serum standardized with one of these calibrators. The wave length chosen is usually close to the wave length of maximum of absorption of the reduced form of the colour reagent. When the colour reagent is nitro-blue tetrazolium the preferred wave length is 550 nm. In an embodiment of the method, the sample and the above described first and second liquid reagents are mixed simultaneously. It is also possible to mix first the above described first and second liquid reagents and subsequently to add the sample to the mixture of reagents. The second liquid is then preferably used in such an amount as to adjust the pH of the reaction mixture between about 9 and about 11, preferably between about 10 and about 11. The use of the set of reagents of the invention has the advantage, compared to commercial sets of reagents comprising a tablet, that mixing of reagents can be quickly conducted and completely automated. The present invention will be further illustrated by the following examples. Example 1: Preparation of a set of reagents A set of first and second liquid reagents according to the invention was prepared with the following composition: First liquid reagent R1: 1.2 mmol/l nitro-blue tetrazolium (NBT) 4000 U/l uricase of Arthrobacter protophormiae (Boehringer Mannheim) 80 mmol/l isotridecylpoly(ethyleneglycolether)8 (Genapol X-80, Boehringer Mannheim) 12 mmol/l sodium cholate 100 mmol/l potassium phosphate buffer pH 7.5 100 mmol/l potassium chloride Second liquid reagent R2: 1.5 mol/l potassium carbonate buffer pH 10.4 Example 2: Study of the stability of the set of reagents 1) Stability of under conditions of storage. This stability was studied, by measuring the absorption at a wave length of 550 nm and determining the uricase activity (by spectrometrically following, at a wavelength of 293 nm and a temperature of 25°C, the transformation of uric acid in presence of catalase, as recommended by Boehringer Mannheim), for a sample of freshly prepared first liquid reagent R1, a sample of first liquid reagent R1 after one year of storage at a temperature between 2 and 8 °C and a sample of first liquid reagent R1 after one year of storage at a temperature between 24 and 26 °C, and by performing the method described in Example 3 under point 1) b) -mode C- with the above samples of reagent R1, and samples of reagent R2 stored in the same conditions, and thereby measuring the photometric linear response maximum concentration and the calibration factor. Two control serum lots and one calibrator lot ( human serum containing glycated albumin, standardized with glycated polylysine) were used as samples. Results are shown in Table 1, hereafter. fresh 1 year 2-8°C 1 year 24-26°C Absorption at 550 nm 0.009 0.007 0.024 % uricase activity compared with freshly prepared R1 100 105 77 Linear response maximum concentration (µmol/l) 1251 1591 1568 Calibration factor 7754 7896 7450 Control serum lot with a specified fructosamine concentration of 286 µmol/l 266 265 280 Control serum lot with a specified fructosamine concentration of 551 µmol/l 532 519 554 Calibrator lot with a specified fructosamine concentration of 479 µmol/l 478 484 493 As can be seen on this table, none of the measured parameters shows a significant variation after one year of storage at 2-8 °C. After one year of storage at 24-26 °C, the calibration factor and the photometric linear response maximum concentration are stable, whereas the uricase activity shows a slight decrease and the absorption a slight increase: these minor changes do not affect the practical use of this reagent in tests, uricase being used in excess and the slight change of the absorption value having no effect on the kinetic measurement. 2) Stability under conditions of use. Conditions of use were simulated by keeping first liquid reagent R1 in a bottle with a small opening allowing air exchange. Three of the above coefficients were measured, immediately after first opening of the bottle and after 8 weeks of storage at a temperature between 4 and 8 °C. Results are shown in Table 2, hereafter. Freshly opened 8 weeks of use Absorption at 550 nm 0.006 0.007 Linear response maximum concentration (µmol/l) 1772 1769 Calibration factor 7508 7434 None of the measured parameters shows a significant variation after 8 weeks of storage under these conditions. The first liquid reagent, immediately after first opening of the bottle and after 8 weeks of storage at a temperature between 2 and 8 °C, was used for measuring the fructosamine content of different plasma samples containing between 120 and 750 µmol/l of fructosamine, according to the method described in Example 3 1) b), mode C. 47 measurements were made for each of these solutions and the results were correlated. The regression straight line equation is: Y= 1.00 X + 3.00 ( X and Y being respectively the values measured for the first liquid reagent, immediately after first opening of the bottle and after 8 weeks of storage at a temperature between 2 and 8 °C).There is no significant difference between these two solutions with a confidence interval of 99.5%. The above described results show the excellent stability of the set of reagents of the invention under conditions of storage and of use. Example 3: Comparison between the method of the present invention and a commercially available method of the prior art. Three different modes of the method of the present invention were used for measuring the fructosamine content of plasma samples containing between 50 and 650 µmol/l of fructosamine and results were correlated to those obtained with the commercially available Fructosamine Plus method, used as reference method. 1) Description of the tests All tests were carried out in a Cobas Mira analyser, using a reaction temperature of 37°C and a spectrometric measurement at 550 nm. a) Fructosamine Plus method A commercial kit Fructosamine Plus (Roche) was used. One tablet containing NBT and uricase was dissolved in 20 ml of the buffer of pH 10.4 containing detergent. 200 µl of the reagent mixture were simultaneously mixed with 10 µl of the sample and 30 µl of water. The reaction rate was measured between 8.3 and 13.8 minutes. b) Method of the invention Mode A: 7 ml of the first liquid reagent (0.82 mmol/l NBT; 70 mmol/l potassium chloride; 70 mmol/l potassium phosphate buffer pH 7.5; 56 mmol/l Genapol X-80; 5.6 mmol/l sodium cholate; 4000 U/l uricase of Arthrobacter protophormiae) were mixed before use with 3 ml of the second liquid reagent (500 mmol/l potassium carbonate buffer pH 10.4) 200 µl of the reagent mixture were simultaneously mixed with 10 µl of the sample and 30 µl of water. The reaction rate was measured between 8.3 and 13.8 minutes. Mode B: The first and second liquid reagents, which had the same composition as in mode A, were used separately on the analyser. 140 µl of the first liquid reagent were simultaneously mixed with 60 µl of the second liquid reagent and 10 µl of the sample. The reaction rate was measured between 8.3 and 13.8 minutes. Mode C: The first liquid reagent (1.2 mmol/l NBT; 100 mmol/l potassium chloride; 100 mmol/l potassium phosphate buffer pH 7.5; 80 mmol/l Genapol X-80; 12 mmol/l sodium cholate; 4000 U/l uricase of Arthrobacter protophormiae) and the second liquid reagent (1500 mmol/l potassium carbonate buffer pH 10.4) were used separately on the analyser. 100 µl of the first liquid reagent were simultaneously mixed with 10 µl of the sample and 40 µl of water. After 3.3 min the reaction was started by adding 20 µl of the second reagent and 40 µl of water. The reaction rate was measured between 13.3 and 15.4 minutes. 2) Results Results obtained using the method of the invention above described methods A, B and C were correlated to those obtained using the reference method. The number of measurements made and the slope a and the intercept b of the regression straight line Y= a X + b (Y being the value measured for the method according to the invention and X being the value measured for the reference method), as well as the Spearman rank correlation coefficient are shown in table 3 hereafter. The above results show that each of the above described modes of the method of the invention has a very high degree of correlation with the reference method. The very stable set of reagents according to the invention will therefore also allow excellent clinical results.

Claims: Method of determining the fructosamine content in a blood sample or a sample derived from blood, by treating the sample with a colour reagent and measuring the induced change of colour, whereby non specifically reducing and/or turbidity-causing components of the sample are removed during the colour reaction, wherein that a first liquid reagent having a pH between 6 and 9 and containing an appropriate non reducing buffering agent, a colour reagent, which is reduced by fructosamine under alkaline pH conditions, thereby changing the colour, and reagents for removing non specifically reducing and/or turbidity-causing components of the sample, is mixed with the second liquid containing a basic buffering agent and, simultaneously or subsequently, the colour reaction is started by adding the sample, whereby the second liquid reagent is used in such an amount as to adjust the pH of the reaction mixture between 9 and 12.

Inventors: VORBERG EWALD, VORBERG, EWALD
Applicants: HOFFMANN LA ROCHE, F. HOFFMANN-LA ROCHE AG
Application year: 1994
Main classification: G01N33
IPC codes: G01N1, G01N33